Methods of treating joint diseases, disorders, and conditions with tissue inhibitors of matrix metalloproteinases

ABSTRACT

Methods for treating joint diseases, disorders, and conditions, such as osteoarthritis, with a composition containing a therapeutically effective amount of tissue inhibitors of matrix metalloproteinases (TIMPs) are provided. The composition can contain TIMPs as part of amniotic fluid, or the TIMPs can be isolated from another source or recombinantly produced. The methods are particularly useful for treating osteoarthritis associated with degradation of articular cartilage by proteases. Also provided are methods of lubricating a joint, such as an osteoarthritic joint.

CROSS REFERENCE TO RELATED APPLICATION

This application is entitled to priority pursuant to 35 U.S.C. § 119(e)to U.S. Provisional Patent Application No. 62/140,909, filed Mar. 31,2015, which is herein incorporated by reference in its entirety.

BACKGROUND OF THE INVENTION

Viscosupplements are viscous substances that can function as a lubricantand shock absorber for joints, and are often used to treat conditions ofthe human musculoskeletal system. Many viscosupplements typically usedfor treating inflammatory conditions of the human musculoskeletal systemare produced via bacterial culturing, or by a process of extraction fromtissues of a different species, such as rooster combs, which is thefleshy growth on top of the rooster's head.

The primary viscous, protecting, nutritional and lubricating fluid ofthe human musculoskeletal system responsible for maintaining healthy,articulating joints is synovial fluid. Synovial fluid is secreted by thesynovial membrane, which is the soft tissue found between the articularcapsule (joint capsule) and joint cavity. In native joints, synovialfluid functions as a biomechanical lubricant, lowering the friction andwear of articulating cartilage in joints. Synovial fluid lubricatingmacromolecules, including hyaluronic acid and proteoglycan 4 (PRG4), aresecreted by synoviocytes in the synovial membrane lining the joint andchondrocytes in the cartilage of the joint, and are concentrated insynovial fluid due to the retaining property of the semi-permeablesynovial membrane.

Currently available viscosupplements for the treatment of inflammatoryconditions of the human musculoskeletal system are comprised ofessentially a single component, hyaluronic acid (HA). Considering thecomplexity of naturally occurring synovial fluid, and the complexbiological system necessary for maintaining the health and properfunctioning of articulating joints, pure HA is not an ideal treatmentfor joints at least for physiological reasons.

Furthermore, the clinical record of currently available HA basedviscosupplements is poor. That record was recently reviewed by theAmerican Academy of Orthopedic Surgeons (AAOS), and in June, 2013, theAAOS issued clinical practice guidelines to physicians, whichrecommended against using HA for patients with symptomaticosteoarthritis (OA) of the knee based on supporting evidence fromseveral high-quality research studies that met the inclusion criteria.

The AAOS's clinical practice guidelines are based on some of the bestpeer reviewed study evidence available. According to the AAOS's website,currently published studies do not show a clinically effective responsefor HA injections based on minimal clinically important improvements(MCIIs). Some peer reviewers were critical of the AAOS' findings andrecommendation, especially in light of the important clinical practiceimplications, and highlighted prior systematic reviews supporting theuse of HA. However, these reviews were analyzed and found to haveseveral flaws. For example, most did not address the issues ofpublication bias, between-study heterogeneity, and clinical significancein determining final recommendations.

Inflammation of articulating surfaces of the musculoskeletal system isone of the most common medical complaints. Although the exact causes forpainful knee, hip, shoulder, facet, ankle, and wrist joints may bedifficult to ascertain and in many cases are unknown, it is understoodthat degenerative damage, especially cartilage damage, plays a centralrole in the pathogenic mechanism leading to this disorder. Currenttreatment modalities include pharmacological treatments, physiotherapy,viscosupplement injections, corticosteroid injections and, at theterminus of a continuum of care for joint pain, surgical replacement ofthe joint. According to the Centers for Disease Control (CDC), nearlyone in two people are projected to develop symptomatic kneeosteoarthritis by age 85 years; two in three people who are obese areprojected to develop symptomatic knee osteoarthritis in their lifetime;and one in four people are projected to develop hip arthritis in theirlifetime. Also, according to the CDC, an estimated 52.5 million adultsin the United Stated reported being told by a doctor that they have someform of arthritis, rheumatoid arthritis, gout, lupus, or fibromyalgia.

Moreover, by 2030, the number of Americans aged 18 years or older whoare projected to have doctor-diagnosed arthritis is 67 million.

Present pharmacological treatments for such joint inflammation includethe use of non-steroidal anti-inflammatory drugs (NSAIDs), such asnaproxen, ibuprofen, etc., and drugs of the cyclooxygenase-2 inhibitorgroup like celecoxib, as well as other drugs including glucosamine,chondroitin, and opiates. Present non-pharmacological treatments includehot or cold packs around the inflamed joint; anaerobic exercises, suchas resistance training; suggestion of weight loss or use of a crutch;use of a brace, particularly for the patella; and correction of jointtiling or misalignment.

However, many of the pharmacological and non-pharmacological treatmentsemployed for treating joint inflammation suffer from several drawbacks.For example, corticosteroid injections are one of the most commonanti-inflammatory treatments for joint pain, and it carries many risksincluding deteriorating articulating cartilage in the joint if overused,atrophy of subcutaneous fat, and nerve inflammation. There is, as aresult, growing interest in the development of novel technologies torepair or regenerate the painful, degenerated articulatingmusculoskeletal bone and cartilage system.

Accordingly, there exists a need in the art for improved methods andcompositions for treating inflammation of the human musculoskeletalsystem, and particularly of joint diseases, disorders, and conditions,such as osteoarthritis. Preferably, such improved methods andcompositions provide the requisite lubrication, cushioning, andprotection of the joint, but more closely resemble the composition ofsynovial fluid found in healthy articulating joints as compared to knownviscosupplements.

BRIEF SUMMARY OF THE INVENTION

The invention satisfies this need by providing methods of treating jointdiseases, disorders, and conditions with a composition comprising ahuman amniotic fluid comprising a therapeutically effective amount oftissue inhibitors of matrix metalloproteinases, which can inhibitprotease activity that contributes to cartilage degradation anddegeneration in the joint, thus facilitating treatment of the jointdisease, disorder, or condition.

In one general aspect, the invention relates to methods of treatingjoint diseases, disorders, and conditions with compositions comprisingan amniotic fluid comprising a therapeutically effective amount oftissue inhibitors of matrix metalloproteinases.

In one embodiment, a method of treating a joint disease, disorder, orcondition in a subject comprises injecting a composition comprising anamniotic fluid comprising a therapeutically effective amount of tissueinhibitors of matrix metalloproteinases (TIMPs) into the joint of thesubject, thereby treating the joint disease, disorder, or condition.

In a preferred embodiment, the joint is a synovial joint.

In another embodiment, the joint disease, disorder, or condition isosteoarthritis, such as osteoarthritis associated with degradation ofarticular cartilage by proteases.

In particular embodiments of the invention, the amniotic fluid isprocessed by removing water to enrich the amniotic fluid for endogenousTIMPs.

In other particular embodiments of the invention, the amniotic fluid isobtained from a donor during a period of gestation when natural levelsof TIMPs in the amniotic fluid are relatively high, such as between week28 and week 37 of gestation.

In another general aspect, the invention provides a method oflubricating an osteoarthritic joint in a subject, wherein theosteoarthritis of the joint is associated with degradation of articularcartilage by proteases, the method comprising injecting into the joint acomposition comprising an amniotic fluid comprising a therapeuticallyeffective amount of tissue inhibitors of matrix metalloproteinases,thereby lubricating the osteoarthritic joint.

In another general aspect, the invention provides a method of treating ajoint disease, disorder, or condition in a subject, the methodcomprising injecting a composition comprising a therapeuticallyeffective amount of tissue inhibitors of matrix metalloproteinases(TIMPs) into the joint of the subject, thereby treating the jointdisease, disorder, or condition.

In yet another general aspect, the invention provides a compositioncomprising an amniotic fluid comprising a therapeutically effectiveamount of tissue inhibitors of matrix metalloproteinases (TIMPs),wherein at least a portion of the TIMPs are exogenously added. Theexogenous TIMPs can be recombinantly produced and/or isolated orpurified from a natural source.

Other aspects, features and advantages of the invention will be apparentfrom the following disclosure, including the detailed description of theinvention and its preferred embodiments and the appended claims.

DETAILED DESCRIPTION OF THE INVENTION

Various publications, articles and patents are cited or described in thebackground and throughout the specification; each of these references isherein incorporated by reference in its entirety. Discussion ofdocuments, acts, materials, devices, articles or the like which has beenincluded in the present specification is for the purpose of providingcontext for the invention. Such discussion is not an admission that anyor all of these matters form part of the prior art with respect to anyinventions disclosed or claimed.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood to one of ordinary skill inthe art to which this invention pertains. In this application, certainterms are used, which shall have the meanings as set in thespecification. It must be noted that as used herein and in the appendedclaims, the singular forms “a,” “an,” and “the” include plural referenceunless the context clearly dictates otherwise.

It is now discovered that compositions comprising human amniotic fluidcomprising a therapeutically effective amount of tissue inhibitors ofmatrix metalloproteinases are effective in treating joint diseases,disorders, and conditions, such as osteoarthritis, and particularlyosteoarthritis associated with degradation of articular cartilage byproteases. Such compositions are also useful for lubricating anosteoarthritic joint to provide, for example, pain free joint movement.

As used herein, the term “subject” means any animal, and preferably amammal. Examples of mammals include, but are not limited to, cows,horses, sheep, pigs, cats, dogs, mice, rats, rabbits, guinea pigs,monkeys, humans etc., more preferably a human.

As used herein, the term “donor” refers to a female subject from whomamniotic fluid will be or has been obtained. The donor can be anyanimal, preferably a mammal, e.g., bovine, human, or porcine, and ismost preferably a human.

“Synovial fluid,” as used herein, refers to the viscous fluid found inthe cavities of synovial joints. Synovial fluid is also referred to as“joint fluid.” Synovial fluid is secreted by the synovial membrane,which is the inner membrane lining a synovial joint. The primary role ofsynovial fluid is to reduce friction between articular cartilage ofsynovial joints during movement. Synovial fluid has other functions,including, but not limited to, reducing joint friction throughlubrication, shock absorption, and nutrient and waste transportation.

As used herein, the term “joint disease, disorder, or condition” refersto any disease, disorder, or condition characterized by jointinflammation, cartilage inflammation in a joint, and/or cartilagedegradation in a joint. Examples of joint diseases, disorders, orconditions include, but are not limited to, joint inflammation, such asarthritis, particularly osteoarthritis and rheumatoid arthritis; andinflammation of the cartilage, such as chondromalacia patellae.

Examples of joints include synovial joints, cartilaginous joints, andfibrous joints. According to embodiments of the invention, jointdiseases, disorders, and conditions can affect synovial and/orcartilaginous joints. “Synovial joints” refer to the most common andmovable type of joint in the body of a mammal. Synovial joints includehinge joints (e.g., elbow and knee), pivot joints (e.g., atlas and axisbones at the top of the neck), ball and socket joints (e.g., hip),saddle joints (e.g., carpometacarpal joint of the thumb), condyloidjoints (e.g., wrist, metacarpophalangeal joints, metatarsophalangealjoint), and gliding joints (e.g., intercarpal joints in the wrist).“Cartilaginous joints” are joints connected entirely by cartilage, suchas the manubrio-sternal joint (sternum) and amphiarthoses joints, suchas intervertebral discs.

In certain embodiments of the invention, the joint disease, disorder, orcondition to be treated affects a synovial joint. In other embodimentsof the invention, the joint disease, disorder, or condition to betreated affects a cartilagnious joint.

Non-limiting examples of synovial joints include, but are not limitedto, knee, wrist, shoulder, hip, elbow, facet, carpal-metacarpal, andtarsal/metatarsal joints.

In a preferred embodiment of the invention, the joint disease, disorder,or condition to be treated is osteoarthritis. As used herein,“osteoarthritis” refers to a form of arthritis occurring in synovialjoints. It is usually a chronic condition, and occurs when theprotective cartilage, known as articular cartilage, on the ends of bonesthat come together to form joints wears down and/or is degraded.“Articular cartilage” refers to the tissue at the ends of bones injoints, which provides frictionless contact between the bones in a jointduring movement. Articular cartilage is composed of two majorcomponents: collagen and proteoglycans. The breakdown of cartilage insynovial joints can be caused by a number of factors including, but notlimited to, proteases, aging, being overweight, and genetic defects incartilage formation. Proteases known to cause degradation of cartilageinclude, but are not limited to, matrix metalloproteinases.

In a particularly preferred embodiment of the invention, the jointdisease, disorder, or condition to be treated is osteoarthritisassociated with degradation of articular cartilage by proteases. As usedherein, “osteoarthritis associated with degradation of articularcartilage by proteases” is intended to specifically refer toosteoarthritis, wherein protease degradation of articular cartilagecauses, exacerbates, or contributes to the onset of the osteoarthritis.Proteases that cause, exacerbate, or contribute to the onset of theosteoarthritis include matrix metalloproteinases, and more particularlymatrix metalloproteinases that degrade collagen.

The terms “matrix metalloproteinases” and “MMPs” are intended to referto a family of zinc-dependent endopeptidases. MMPs play a role in manybiological processes, including degradation of articular cartilage. MMPsare first produced and released in an inactive form known as the“pro-MMP” form. Inactive pro-MMPs are subsequently activated byproteases that cleave off a portion of the protein. Examples of MMPsinclude, but are not limited to, MMP-1, MMP-2, MMP-3, MMP-4, MMP-5,MMP-6, MMP-7, MMP-8, and MMP-9. Although the specific natural substratesfor each of the MMPs varies, MMPs are generally capable of degradingextracellular matrix proteins, such as collagen, gelatin, proteoglycans,etc. The MMPs that are currently known to specifically play a role incartilage extracellular matrix degradation include, but are not limitedto, the collagenases (MMP-1, MMP-8, MMP-13), the stromelysins (MMP-3,MMP-10, MMP-11), the gelatinases (MMP-2, MMP-9), matrilysin (MMP-7), andmembrane type MMPs, in particular MMP-14, which can also act as acollagenase.

As used herein, the terms “tissue inhibitor of matrixmetalloproteinases” and “TIMPs” refer to a family of protease inhibitorsthat modulate the activity of matrix metalloproteinases. To date, it isknown that the human genome encodes four TIMPs: TIMP1, TIMP2, TIMP3, andTIMP4. TIMPs modulate matrix metalloproteinases by inhibiting theactivity of activated MMPs, or by blocking the activation of pro-MMPs.According to embodiments of the invention, an amniotic fluid comprisinga therapeutically effective amount of TIMPs can affect the activity ofMMPs in a joint by inhibiting the activity of activated MMPs, or byblocking the activation of pro-MMPs, thereby reducing the breakdown ofcartilage by MMPs in the joint.

As used herein, the terms “treat,” “treating,” and “treatment” are allintended to refer to an amelioration or reversal of at least onemeasurable physical parameter related to a joint disease, disorder, orcondition, which is not necessarily discernible in the subject, but canbe discernible in the subject. The terms “treat,” “treating,” and“treatment,” can also refer to preventing the progression, or at leastslowing down the progression of the joint disease, disorder, orcondition. In one embodiment, “treat,” “treating,” and “treatment” referto a reduction or complete alleviation of pain associated with the jointdisease, disorder, or condition. In another embodiment, “treat,”“treating,” and “treatment” refer to a reduction of joint inflammation.In yet another embodiment, “treat,” “treating,” and “treatment” refer toinhibiting or reducing the degradation of cartilage in a joint, such asarticular cartilage in a synovial joint. And in yet another embodiment,“treat,” “treating,” and “treatment” refer to an alleviation of one ormore symptoms associated with the joint disease, disorder, or condition,such as joint pain, joint swelling, joint stiffness, inflammation,difficulty in joint movement, and reduced range of motion.

The invention provides methods of treating joint diseases, disorders,and conditions in a subject.

In one general aspect, the invention provides a method of treating jointdiseases, disorders, and conditions in a subject, the method comprisinginjecting into the joint a composition comprising an amniotic fluidcomprising a therapeutically effective amount of tissue inhibitors ofmatrix metalloproteinases (TIMPs). TIMPs can inhibit the activity ofproteases that increase cartilage degradation in a joint, particularlyMMPs, thereby facilitating treatment of the joint disease, disorder, orcondition.

According to embodiments of the invention, an amniotic fluid can be anyamniotic fluid from a mammal including, but not limited to, bovineamniotic fluid, porcine amniotic fluid, and human amniotic fluid, and ismost preferably human amniotic fluid.

Amniotic fluid naturally contains TIMPs, as well as many otherregenerative components that are efficacious for treating jointdiseases, disorders, and conditions, such as growth factors, stem cells,protease inhibitors, etc. Moreover, certain compositions used with theinvention contain many of the same components as the synovial fluidfound in healthy joints, and are thus particularly suited for covering,lubricated, protecting, and relieving joint inflammation and joint painwhen injected into the joint. Accordingly, an advantage of providing atherapeutically effective amount of TIMPs in an amniotic fluid is thatthe other components naturally present in the amniotic fluid can furtherenhance treatment of joint diseases, disorders, and conditions, andfurther alleviate the symptoms thereof.

According to embodiments of the invention, the amniotic fluid of thecomposition comprises a therapeutically effective amount of tissueinhibitors of matrix metalloproteinases. The term “therapeuticallyeffective amount” refers to an amount of an active ingredient orcomponent that elicits the desired biological or medicinal response in asubject. As used herein with reference to tissue inhibitors of matrixmetalloproteinases (TIMPs), a therapeutically effective amount means anamount of TIMPs that results in treatment of a joint disease, disorder,or condition; prevents or slows the progression of the joint disease,disorder, or condition; reduces or completely alleviates pain associatedwith the joint disease, disorder, or condition; reduces or preventscollagen degradation by proteases in the joint; reduces jointinflammation; or alleviates one or more symptoms associated with thejoint disease, disorder, or condition, such as joint pain, jointswelling, joint stiffness, inflammation, reduced range of motion, anddifficulty in joint movement.

One of ordinary skill in the art will recognize that the therapeuticallyeffective amount of TIMPs to be used in the invention can vary with manyfactors, such as the particular subject, (e.g., age, diet, health,etc.); particular joint disease, disorder, or condition; severity of thejoint disease, disorder, or condition, and any underlying complicationsor conditions in the subject that may have caused or contributed to theonset of the joint disease, disorder, or condition. For example, manyosteoarthritis patients have one or more underlying complications orconditions that contribute to the onset and progression ofosteoarthritis, such as obesity, etc.

According to embodiments of the invention, a therapeutically effectiveamount of TIMPs in the amniotic fluid can be provided by the naturallevel of TIMPs endogenously present in the amniotic fluid. Thus, anamniotic fluid collected from a donor can be used in a composition forinjection into a joint without further processing or manipulation. Incertain embodiments of the invention, the amniotic fluid is collectedfrom the donor at a point during gestation when TIMP levels aretypically elevated.

During gestation of an embryo, the body creates a placental sac made ofamnion and chorion tissues surrounding the fetus and containing amnioticfluid to both protect and nourish the fetus. A purpose of TIMPs inamniotic fluid is to prevent the amnion and chorion tissues from beingprematurely degraded by proteases, as such degradation would causepremature rupture of the placental sac and pre-term birth of the fetus.However, once the pregnancy has reached full term, TIMP levels in theamniotic fluid typically abruptly decrease, which allows for degradationof the amnion and chorion tissues, release of the amniotic fluid, andsubsequent birth of the fetus. Thus, TIMP levels in amniotic fluid areusually at their highest during the late pre-term period of pregnancy,which prevents degradation of the amnion and chorion tissues, thusmaintaining the integrity of the placental sac.

According to embodiments of the invention, the amniotic fluid, such ashuman amniotic fluid, can be obtained from a donor during gestation at apoint when TIMP levels are typically near or at their maximum. As knownby one of ordinary skill in the art, the gestation period in humanfemales is divided into three trimesters: the first trimester (weeks onethrough twelve), the second trimester (weeks thirteen throughtwenty-seven), and the third trimester (weeks twenty-eight to birth).Gestation in human females is also characterized according to terms,i.e., specific periods of gestation during which birth occurs: pre-term(before 37 weeks of gestation), early term (37 weeks to 38 weeks, 6 daysof gestation), full term (39 weeks to 40 weeks, 6 days of gestation),and late term (41 weeks to 41 weeks 6 days of gestation). TIMP levelshave been demonstrated to fall rapidly in amniotic fluid at birth.Therefore, in a preferred embodiment, the amniotic fluid is obtainedfrom a human donor at a point just prior to birth.

According to embodiments of the invention, human amniotic fluid for usein the invention can be obtained from a human female donor. Preferably,the human amniotic fluid is obtained at the time of Caesarean sectiondelivery. When the Caesarean section delivery is elective, collection ofthe amniotic fluid can occur during the late pre-term period ofpregnancy, i.e., during weeks 28 to 37 of gestation, and preferablycloser to week 37 of gestation, when TIMP levels are typically at ornear their maximum. For example, the amniotic fluid can be obtainedduring week 28, 29, 30, 31, 32, 33, 34, 35, 36, or 37 of gestation.

In a particularly preferred embodiment, amniotic fluid is obtained froma female donor undergoing a Caesarean section procedure, wherein theCaesarean section procedure takes place at a point during gestation nearfull term. Amniotic fluid obtained from a Caesarean section donor nearterm typically has a high concentration of TIMPs. As used herein, thephrase “near full term” refers to weeks 33-37 of gestation, such as week33, 34, 35, 36, or 37 of gestation.

According to embodiments of the invention, the concentration of TIMPs inthe amniotic fluid can be determined by testing a sample of amnioticfluid obtained at the time of Caesarean section, or by testing a samplepreviously obtained via amniocentesis, if desired. Any method known inthe art in view of the present disclosure can be used to determine theconcentration of TIMPs, such enzyme-linked immunoabsorbent assay(ELISA), Bradford assay, electrophoresis techniques (e.g., SDS-PAGE,Western blot), etc. For example, the concentration of TIMPs can bedetermined by testing a sample of amniotic fluid obtained byamniocentesis or at the time of the Caesarean section delivery.

According to embodiments of the invention, a composition used in themethods described herein can further comprise a cryoprotectant. Anycryoprotectant suitable for pharmaceutical use known to those skilled inthe art in view of the present disclosure can be used in the compositionof the present invention. Examples of cryoprotectants that can be usedinclude, but are not limited to, dimethyl sulfoxide (DMSO), sucrose,glycerol, glucose, and any other sugars, e.g., monosaccharides ordisaccharides, alcohols and penetrating agents, or combinations thereof,routinely used as cryoprotectants by those skilled in the art, whichwill be known to those skilled in the art in view of the presentdisclosure.

In some embodiments of the invention, the composition is cryopreservedprior to injection into a joint. The cryopreserved composition can bewarmed prior to injection to a temperature in a range of about 15° C. to25° C., such as 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25° C.

In particular embodiments of the invention, the amniotic fluid is aprocessed amniotic fluid, such as a processed human amniotic fluid. Theterm “unprocessed,” when used with respect to an amniotic fluid, refersto an amniotic fluid that has not been treated, such that it hassubstantially the same composition, i.e., same components in the sameamounts, as the native amniotic fluid found in vivo. The term“processed,” when used with respect to an amniotic fluid, means that theamniotic fluid has been treated or manipulated in some way after beingcollected from the donor, such that it has an altered composition, i.e.,same components in different amounts or different components, ascompared to the native amniotic fluid found in vivo.

According to embodiments of the invention, a processed amniotic fluidcan be prepared from an unprocessed amniotic fluid. For example, aprocessed amniotic fluid is prepared from an unprocessed amniotic fluidby concentrating the amniotic fluid that is collected from a donor toincrease the concentration of TIMPs and/or other components naturallypresent in the amniotic fluid.

In a preferred embodiment, the processed amniotic fluid is prepared byconcentrating an amniotic fluid obtained from a donor by removing atleast water to enrich the amniotic fluid for endogenous TIMPs. Theamniotic fluid can be further processed such that the substantially allof the endogenous components of the amniotic fluid with the exception ofTIMPs are removed from the amniotic fluid. According to embodiments ofthe invention, a processed amniotic fluid can comprise about 10% toabout 100% (w/v) TIMPs, such as, for example, 10%, 20%, 30%, 40%, 50%,60%, 70%, 80%, 90%, 95%, 99%, or 100% (w/v) TIMPs.

However, the invention is not limited to administering TIMPs as acomponent of an amniotic fluid, and embodiments of the invention alsorelate to administering compositions comprising a therapeuticallyeffective amount of TIMPs.

Thus, in yet another general aspect of the invention, a method oftreating a joint disease, disorder, or condition in a subject comprisesinjecting a composition comprising a therapeutically effective amount oftissue inhibitors of matrix metalloproteinases (TIMPs) into the joint ofthe subject, thereby treating the joint disease, disorder, or condition.The TIMPs can be obtained from an amniotic fluid, or isolated orpurified from another natural source. The TIMPs can also berecombinantly produced by any method known in the art. Recombinantlyproduced TIMPs include, but are not limited to, human recombinant TIMPs.

Embodiments of the invention also relate to harvesting TIMPs fromamniotic fluid obtained from the donor, preferably amniotic fluidobtained from the donor at a point during gestation when the level ofTIMPs is at or near the maximum. TIMPs harvested from the amniotic fluidcan be purified and used to supplement a composition comprising amnioticfluid prior to injection of the composition into a joint. TIMPsharvested from the amniotic fluid can also be purified from the otheramniotic fluid components, and the purified TIMPs can be administered ina composition.

According to embodiments of the invention, a composition is injectedinto a joint. Any of the compositions described herein comprising atherapeutically effective amount of TIMPs can be injected into a joint.Preferably, the composition is injected into a synovial joint, and ismore preferably injected into the intra-articular space of a synovialjoint. The intra-articular space refers to the space inside of a jointbetween two bones that is usually contained by the articular capsule.The articular capsule, also referred to as the joint capsule, is theenvelope surrounding a synovial joint.

In particularly preferred embodiments, the invention relates to a methodof treating osteoarthritis of a synovial joint, comprising injecting acomposition comprising an amniotic fluid comprising a therapeuticallyeffective amount of tissue inhibitors of matrix metalloproteinases intothe synovial joint. Preferably, the osteoarthritis is associated withdegradation of articular cartilage by proteases. According toembodiments of the invention, the synovial joint can be any synovialjoint, such as a knee, wrist, shoulder, hip, elbow, or neck joint. In apreferred embodiment, the synovial joint is a knee joint.

Embodiments of the invention also relate to methods of lubricating ajoint, such as an osteoarthritic joint, using the compositions describedherein, such as a composition comprising an amniotic fluid comprising atherapeutically effective amount of tissue inhibitors of matrixmetalloproteinases. Compositions comprising a therapeutically effectiveamount of TIMPs, such as recombinantly produced TIMPs, or TIMPspurified/isolated from amniotic fluid or another natural source can alsobe used for joint lubrication. Preferably, the joint to be lubricated isa synovial joint, and is more preferably a knee joint. A method oflubricating a joint according to the invention can be used to providepain-free joint movement to the subject, facilitate joint movement,and/or prevent bone wear and tear in a joint.

As used herein, an “osteoarthritic joint” refers to a synovial jointaffected by osteoarthritis. As used herein, “lubricating a joint” refersto supplementing the amount of synovial fluid present in the joint, orreplacing synovial fluid in a joint to increase joint movement and/orprevent bone wear and tear in the joint. Lubricating a joint with acomposition comprising a human amniotic fluid comprising atherapeutically effective amount of TIMPs, or a composition comprising atherapeutically effective amount of TIMPs, has the advantage ofinhibiting proteases in the joint, thereby also preventing or reducingthe breakdown of collagen in the joint while simultaneously replacing orsupplementing the synovial fluid in the joint to improve joint movement.

According to embodiments of the invention, a composition used in thetreatment of a joint disease, disorder, or condition, or lubrication ofa joint, can be used in combination with another treatment including,but not limited to, general exercise; strengthening exercises; walkingaids such as canes or crutches; wheeled walkers or frames; knee bracing;orthotics; non-steroidal anti-inflammatory drugs (NSAIDs); thermaltreatments including heat therapy; cryotherapy; transcutaneouselectrical nerve stimulation; and acupuncture.

According to embodiments of the invention, a composition can be injectedinto a joint once a day or multiple times a day. The composition canalso be injected according to a specified dosing regimen, e.g., once aweek for a period of a few weeks or a few months. The dosing regimen canalso occur over a period of year. An exemplary and nonlimiting exampleof a dosing regimen includes one six injections over the course of ayear. The optimal frequency and duration of injections of thecomposition will depend upon various factors including the particulartype of joint disease, disorder, or condition to be treated, itsunderlying cause, whether or not the composition is being used incombination with another treatment (e.g., general exercise orstrengthening exercises), etc. For example, in some instances, oneinjection will be sufficient to provide a clinically beneficial effect.In other instances, more than one injection, such as one injection everytwo months over the course of the year will be need to realize aclinically beneficial effect. The injection volume can also varydepending upon a variety of factors, such as the particular joint andthe size of the joint. For example, a larger injection volume istypically needed for treatment of a joint disease, disorder, orcondition when the joint is a knee joint as compared to a finger joint.As general guidance, the injection volume can range from about 0.25 mLto about 6 mL, such as 0.25, 0.5, 0.75, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5,4.0, 4.5, 5.0, 5.5, or 6.0 mL. One of ordinary skill in the art will beable to determine the frequency and duration of injections of thecomposition, as well as the appropriate injection volume in view of theabove factors in order to achieve the desired outcome.

In another general aspect, the invention provides a compositioncomprising an amniotic fluid comprising a therapeutically effectiveamount of tissue inhibitors of matrix metalloproteinases (TIMPs),wherein at least a portion of the TIMPs are exogenously added. Theexogenously added TIMPs can be recombinantly produced, and/or isolatedor purified from another natural source. The amniotic fluid can alsocomprises TIMPs that are naturally present in the amniotic fluid. Thecomposition can be used in any of the methods described herein.

Methods of obtaining an amniotic fluid from a donor using theappropriate sterile techniques are well known to those of ordinary skillin the art. One of ordinary skill in the art is also familiar withprocedures for safely and humanely obtaining an amniotic fluid from adonor in an aseptic manner. For example, human amniotic fluid can beobtained from a donor who is undergoing an amniocentesis procedure,Caesarean section delivery, or vaginal birth using a specially designedreceptacle to collect the fluid. Preferably, the amniotic fluid isobtained from a donor undergoing a Caesarean section delivery, and ismore preferably obtained from a donor undergoing a pre-term Caesareansection delivery, i.e., prior to week 37 of gestation. Amniotic fluidobtained from a donor undergoing vaginal birth, or from an amniocentesisprocedure can also be used with a method of the invention, however alarger quantity of amniotic fluid can be obtained from a donorundergoing a Caesarean section delivery, and is thus the preferredmethod for obtaining amniotic fluid. Also, amniotic fluid obtained atthe time of vaginal delivery at term typically has lower levels ofTIMPs.

Preferably, the technique used for harvesting the amniotic fluid shouldsubstantially eliminate, or at least minimize, the presence of red bloodcells in the amniotic fluid. Furthermore, the amniotic fluid used in theinvention should not be cloudy in color, and it should not have anyparticulate matter. Particulate matter can be removed from the amnioticfluid by any method known in the art for removing particulate matterfrom biological samples including, but not limited to, filtration andcentrifugation. Particulate matter can be removed at any time after theamniotic fluid has been collected. Preferably, the particulate materialis removed prior to any other processing or treatment steps.

According to embodiments of the invention, amniotic fluid used in theinvention is procured from a female donor. Informed consent is obtainedfrom the female donor by following guidelines as promulgated by theAmerican Association of Tissue Banks and consistent with guidelinesprovided by the Food and Drug Administration: a federal agency in theDepartment of Health and Human Services established to regulate therelease of new medical products and, finally, if required by anestablished review body of the participating hospitals or institutions.The female donor is informed that she will be subject to risk assessmentto determine if she is qualified as an amniotic fluid donor. She willalso be informed of the tests for the risk assessment. The female donoris further informed that, if she is selected as an amniotic fluid donorbased on the risk assessment, her birth tissues, such as placenta andamniotic fluid, may be collected at birth, tested and processed formedical uses. The informed consent includes consent for risk assessmentand consent for donation of birth tissues and amniotic fluid.

Risk assessment is conducted on the female donor with informed consentto evaluate her risk factors for communicable diseases, such as humanimmunodeficiency virus (HIV), hepatitis B virus (HBV), hepatitis C virus(HCV), cytomegalovirus (CMV), human T-lymphotropic virus (HTLV),syphilis, etc., as is required by regulating bodies. Medical and socialhistories of the female donor, including physical exam record, and/orrisk assessment questionnaire, are reviewed. Those with high riskfactors for the communicable diseases are excluded.

Consent to draw blood at the time of delivery and 1 to 12 monthspost-delivery is obtained from female donors with low risk factors forthe communicable diseases. Screening tests on communicable diseases,such as HIV 1 and 2, HCV, HbCore, syphilis, HTLV I/II, CMV, hepatitis Band C, are conducted by conventional serological tests on the bloodsample obtained at birth. The initial screening tests are preferablycompleted within 7 days after birth. Preferably, the screening tests areconducted again on a second blood sample collected a few monthspost-delivery, to verify the previous screening results and to allow fordetection of communicable disease acquired shortly before birth, but areshown as “negative” on the previous screening tests. The second bloodsample can be collected 1-12 months, preferably 6 months, post birth.

Only female donors with informed consent who are tested negative for thecommunicable diseases are approved as amniotic fluid donors. In apreferred embodiment, only female donors with informed consent who aretested negative for the communicable diseases in both screening testswith the blood sample drawn at birth and the blood sample drawn 6 monthspost-delivery are approved as amniotic fluid donors.

According to embodiments of the invention, the amniotic fluid can betreated in such a way as to promote preservation, lengthen shelf life,etc. These treatments include, but are not limited to, sterilization,e.g., gamma-irradiation; cooling, refrigeration, and freezing; andaddition of one or more preservatives, cryopreservatives, antimicrobialagents, etc.

According to embodiments of the invention, once collected from thedonor, the amniotic fluid can be processed to provide a processedamniotic fluid. Thus, in particular embodiments of the invention, anamniotic fluid of a composition used in a method of the invention is aprocessed amniotic fluid.

In one embodiment, the amniotic fluid is processed to remove water, andoptionally other components from the amniotic fluid. For example, usingan ultrafiltration approach, a semi-permeable container is filled withraw amniotic fluid, and then a pressure gradient is applied across thesemi-permeable membrane using any number of techniques known to thoseskilled in the art including, but not limited to, a high permeabilitydialyzer. As another illustrative example, when employing hemodialysistechniques, an electrolyte solution (dialysate) can be applied on oneside of a membrane, creating a concentration gradient, which causeswater and other non-protein cellular components of the amniotic fluid toflow through the semi-permeable membrane. As yet another illustrativeexample, rapid ultrafiltration approaches can be used. Rapidultrafiltration approaches employ a semi-permeable membrane cylindricalcontainer that rotates constantly in order to avoid filter clogging evenas a pressure gradient is applied to the contained fluid—either fromwithin the container (pushing), or from the opposite side of thesemi-permeable membrane (pulling).

A processed amniotic fluid can be prepared by removing water from theamniotic fluid obtained from a donor by any technique known to those ofordinary skill in the art to provide a processed amniotic fluid. Forexample, substantially all of the water can be removed bylyophilization, etc., or the amount of water can be reduced by vacuumfiltration, etc.

A processed amniotic fluid can also be prepared from an unprocessedamniotic fluid by increasing the concentration of one or more ofproteins, carbohydrates, lipids, and other desirable components in theamniotic fluid using any technique known to those of ordinary skill inthe art in view of the present disclosure. For example, a desirableendogenous component, e.g., TIMPs, can be enriched by filtration orcentrifugation with specific parameters. An exogenous desirablecomponent can also be added to the processed amniotic fluid.

In another embodiment, a processed amniotic fluid has a differentviscosity as compared to the unprocessed amniotic fluid. The amnioticfluid can be further processed so that it has a relatively highviscosity for ease of application and for remaining in the desired areaafter the application. For example, the amniotic fluid can beconcentrated to remove water by centrifugation, lyophilization, vacuumfiltration, etc., which will also concentrate many of the other amnioticfluid components, such as TIMPs, thus increasing their concentration.Increasing the concentration of TIMPs and/or other components in theamniotic fluid can achieve optimal therapeutic concentrations of TIMPs,as well as other amniotic fluid components, which can further enhancetreatment of the joint disease, disorder, or condition.

In one embodiment, a processed amniotic fluid has one or more differentcomponents, such as proteins, lipids, and carbohydrates, as compared tothe unprocessed amniotic fluid.

In another embodiment, a processed amniotic fluid has the samecomponents, such as proteins (e.g., TIMPs), lipids, and carbohydrates,as compared to the unprocessed amniotic fluid, but the components arepresent in different amounts, i.e., in an increased concentration or adecreased concentration.

For example, a processed amniotic fluid comprising a therapeuticallyeffective amount of TIMPs can be an amniotic fluid supplemented withexogenous TIMPs. Supplementing the amniotic fluid collected from thefemale donor with exogenous TIMPs, e.g., purified TIMPs or recombinantlyproduced TIMPs, can increase the effective concentration of TIMPs in theamniotic fluid and further enhance its effects on treating jointdiseases, disorders, and conditions.

In another embodiment, a processed amniotic fluid is prepared from anunprocessed amniotic fluid by removing non-protein nitrogenouscompounds, such as uric acid, urea, creatinine, and nitrogen from theamniotic fluid. These non-protein nitrogenous compounds can be removedby any method known in the art, such as by means of a semi-permeablemembrane or filter, or by dialysis. Other suitable means for removingthese compounds include osmotic, centrifugal, gravitational ormechanical pumping forces.

According to embodiments of the invention, the concentrations of thecomponents found in the unprocessed amniotic fluid, particularlyinorganic constituents, gases, non-protein nitrogenous compounds,proteins, carbohydrates, and lipids, differ from the concentrationsfound in the processed amniotic fluid. The concentration of any oneparticular component in the processed amniotic fluid can be increased,decreased, or unchanged as compared to the concentration in theunprocessed amniotic fluid, independent of any other component. Forexample, the concentration of creatinine in the processed amniotic fluidcan be decreased, whereas the concentration of glucose can be increased,as compared to their respective concentrations in the unprocessedamniotic fluid.

According to embodiments of the invention, a processed amniotic fluidcan comprise an increased concentration of at least one of a firstcomponent selected from the group consisting of proteins, lipids, andcarbohydrates as compared to a concentration in an unprocessed amnioticfluid; and a decreased concentration of at least one of a secondcomponent selected from the group consisting of urea, uric acid, andcreatinine, as compared to a concentration in the unprocessed amnioticfluid.

In one particular embodiment a composition used in a method of theinvention comprises a processed amniotic fluid having increasedconcentrations of proteins as compared to the unprocessed amnioticfluid. According to this embodiment, the total protein concentration canbe increased in the processed amniotic fluid, such that the aggregateprotein content ranges from about 30.25 g/L to about 50.375 g/L. Theconcentrations of at least one of cytokines, such as interleukin (IL)-6,IL-8, IL-1β, and tumor necrosis factor (TNF)-α, can be increased in theprocessed amniotic fluid; and the concentrations of at least one ofglobulins, such as α-globulins, β-globulins, and γ-globulins, can beincreased as compared to their concentrations in the unprocessedamniotic fluid. According to other embodiments of the invention, albumincomprises no less than 60% of the total protein content in the processedamniotic fluid. The concentration of other proteins present in theunprocessed amniotic fluid, such as C-reactive protein, procalcitonin,and/or calprotecin can be present in the processed amniotic fluid, orthey can be eliminated.

In preferred embodiments of the invention, in the processed amnioticfluid, the concentration of α-globulins is at least 12% of the totalprotein concentration, preferably 12% to 18%; the concentration ofβ-globulins is at least 16% of the total protein concentration,preferably 16% to 27%; and the concentration of γ-globulins is at least12% of the total protein concentration, preferably 12% to 18%. In otherpreferred embodiments, in the processed amniotic fluid, theconcentration of IL-6 is at least 329 ng/L, the concentration of IL-8 isat least 421 ng/L, the concentration of IL-1β is at least 3.9 ng/L andthe concentration of TNF-α is at least 11.5 ng/L.

In another particular embodiment, a composition used in a method of theinvention comprises a processed amniotic fluid having increasedconcentrations of lipids as compared to the unprocessed amniotic fluid.According to this embodiment, the concentrations of at least one offatty acids, cholesterol, and phospholipids in the processed amnioticfluid are increased as compared to their respective concentrations inthe unprocessed amniotic fluid. The processed amniotic fluid cancomprise fatty acids at a concentration of about 0.25 g/L to about 6.5g/L; cholesterol at a concentration of about 0.5 g/L to about 9.5 g/L;and/or phospholipids at a concentration of about 0.003 g/L to about 0.3g/L.

In yet another particular embodiment, a composition used in a method ofthe invention comprises a processed amniotic fluid having increasedconcentrations of carbohydrates as compared to the unprocessed amnioticfluid. In this embodiment, the concentration of one or more of glucose,fructose, hyaluronic acid, and lubricin in the processed amniotic fluidis increased as compared to their respective concentrations in theunprocessed amniotic fluid. Lubricin (proteoglycan 4) is a proteoglycanpresent in synovial fluid that acts as lubricant. These carbohydratesfunction to provide lubrication, protection and shock absorption,particularly to inflamed joints. The processed amniotic fluid cancomprise glucose at a concentration of about 600 mg/L to about 900 mg/L;fructose at a concentration of about 48 mg/L to about 59 mg/L;hyaluronic acid at a concentration of about 30 μg/L to about 3600 μg/L;and/or lubricin at a concentration of about 10 μg/ml to about 200 μg/ml.

And in yet another particular embodiment, a composition used in themethod of the invention comprises a processed amniotic fluid havingdecreased concentrations of non-protein nitrogenous compounds ascompared to the unprocessed amniotic fluid. For example, theconcentrations of one or more of urea, uric acid, and creatinine in theprocessed amniotic fluid are decreased as compared to their respectiveconcentrations in the unprocessed amniotic fluid. Preferably, theconcentration of urea is no more than 160 mg/L, the concentration ofuric acid is no more than 80 g/L, and/or the concentration of creatinineis no more than 14 mg/L in the processed amniotic fluid of a compositionused with the invention.

According to embodiments of the present invention, the pH of acomposition ranges from a pH of about 6.0 to a pH of about 8.0, such as6.0, 6.5, 7.0, 7.5, or 8.0. Preferably, the pH ranges from 6.5 to 7.5,and is more preferably 7.0.

In other particular embodiments, compositions used in the methods ofinvention comprise a processed amniotic fluid having optimizedconcentrations of inorganic constituents and optimized partial pressuresof gases, such that the concentrations and partial pressures closelyapproximate the concentrations of these components in the nativesynovial fluid. Such inorganic constituents include sodium, potassium,and chloride, and such gases include oxygen and carbon dioxide.

Other examples of processed amniotic fluids suitable for use in theinvention, and processes for preparing such processed amniotic fluidsare described in U.S. patent application Ser. No. 14/950,186, which isherein incorporated by reference in its entirety.

The compositions used in the invention can further comprise additionalsubstances including pharmaceutically acceptable excipients, such asthickeners, salts, preservatives, colorants, etc.; substances to preventthe growth of microbes, such as antifungal, antibacterial, or antiviralagents; and agents that improve the viscosity or thickness of thecomposition. These additions can be made, provided that they do notcause irritation of the joint, or interfere with the healing propertiesof the amniotic fluid. The compositions of the present invention canalso further comprise one or more pharmaceutically active ingredients,such as an analgesic, an anti-inflammatory agent, an anti-microbialagent, etc.

An illustrative and non-limiting example of an embodiment of acomposition comprising a processed amniotic fluid that can be used in amethod of the invention is shown in Table 1 below. According toembodiments of the invention, the processed amniotic fluid furthercomprises a therapeutically effective amount of tissue inhibitors ofmatrix metalloproteinases.

TABLE 1 Concentrations of inorganic constituents, gases, proteins,carbohydrates, lipids, and non-protein nitrogenous compounds in anembodiment of an amniotic fluid of a composition used in a method of theinvention. Composition Target Ranges Unprocessed post-processingComponent amniotic fluid¹ High Low Discussion and Function InorganicConstituents Sodium 127 mEq/L 148.375 85.625 Stay within prescribedrange Potassium 4.0 mEq/L 48.125 21.875 Increase from about 4.0 mEq/L insource material to a minimum of 21.875 mEq/L Chloride 105 mEq/L 145.7566.25 Stay within prescribed range Calcium 4.0 mEq/L 7.21875 3.28125Stay within prescribed range Magnesium 1.4 mEq/L 2.75 1.25 Stay withinprescribed range Phosphorus 29.0 mg/L 55 25 Increase the amount ofphosphorus to the prescribed range Gases/H+ pH 7.0 7.9375 6.125 In orderto restore the proper acid/base balance in an acidic and arthriticjoint, stay within the prescribed range PO2 2-15 mmHg 78 8 in order toprovide the proper PCO2 57 mmHg 150 34 oxygenation range for use as aviscosupplement in an arthritic joint, increase to the two prescribedranges Protein (Total) 22-31 g/L 50.375 30.25 Increase the amount ofprotein to the prescribed range Albumin 60% 60% Ensure that Albumincomprises no less than 60% of the protein content α-globulins 12% 18%12% Stay within prescribed range β-globulins 16% 27% 16% Stay withinprescribed range γ-globulins 12% 18% 12% Stay within prescribed rangeCytokines IL-6 329 ng/L 329 ng/L No less than the prescribed amount IL-8421 ng/L 421 ng/L No less than the prescribed amount IL-1β 3.9 ng/L 3.9ng/L No less than the prescribed amount TNF-α 11.5 ng/L 11.5 ng/L Noless than the prescribed amount C-reactive protein 5.4 mg/L May bepresent or may be (CRP) eliminated in any embodiment of the inventionProcalcitonin 1.8 μg/L May be present or may be eliminated in anyembodiment of the invention Calprotecin 3425 μg/L May be present or maybe eliminated in any embodiment of the invention Non-Protein Theseby-products of Nitrogen metabolism are reduced in Compounds the finalviscosupplement fluid. Urea 370 mg/L 160 82.5 Reduce quantities suchthat the amounts are no more than the maximum amount prescribed UricAcid 50 mg/L 80 22.5 Reduce quantities such that the amounts are no morethan the maximum amount prescribed Creatinine 28 mg/L 14 8.75 Reducequantities such that the amounts are no more than the maximum amountprescribed Carbohydrates These carbohydrates are important to supplyproper levels of nutrients, lubrication, compression and shearresistance and cushioning to the living musculoskeletal joint Glucose330 mg/L 900 600 Increase quantity to the prescribed ranges Fructose 35mg/L 58.625 48.125 Increase quantity to the prescribed ranges LacticAcid 370-750 mg/L 250 156.25 Reduce quantities such that the amounts areno more than the maximum amount prescribed Pyruvate 8 mg/L 13.4 11Increase quantity to the prescribed ranges Hyaluronan: mean22.67(10.8)/1.1(0.46) 3600 μg/ml 30 μg/L Provides Compression & (SD)μg/L Shear Resistance and is a carrier for surface active phospholipids(SAPL), which are a boundary lubricant in viscosupplement fluids.Increase to levels within the prescribed levels. Lubricin (PRG4) 200μg/ml 10 μg/ml This is a protein which is related to MCF and is likelycarrier for SAPL. Increase to the prescribed levels. Total Lipids 0.48g/L Fatty Acids 0.24 g/L 6.50 g/L 0.25 g/L Increase quantity to theprescribed ranges Cholesterol 0.02 g/L 9.50 g/L 0.50 g/L Increasequantity to the prescribed ranges Phospholipids 0.03 g/L 0.30 mg/ml0.003 g/L Increase quantity to the (Total) prescribed ranges ¹References(24)-(33) were used to determine the quantities of the components in theunprocessed amniotic fluid

Any of the processed amniotic fluids described herein can be used in anyof the methods of the invention including methods of treating jointdiseases, disorders, and conditions, methods of treating osteoarthritisin a synovial joint, methods of treating osteoarthritis associated withdegradation of articular cartilage by proteases, methods of lubricatinga joint, such as an osteoarthritic joint, etc.

Proteases, particularly matrix metalloproteinases (MMPs), are known todegrade the collagen. With respect to joints, MMPs can degrade collagenin the articular cartilage, which causes the cartilage to wear down,leading to various joint conditions including, but not limited toosteoarthritis. Without wishing to be bound by any theories, it isbelieved that TIMPs can inhibit the activity of proteases in joints,particularly MMPs, thereby alleviating joint conditions such asosteoarthritis. Again without wishing to be bound by any theories, it isfurther believed that TIMPs naturally present in amniotic fluid caninhibit the activity of proteases in joints, thereby alleviating jointconditions such as osteoarthritis. Moreover, compositions comprisingamniotic fluid are thought to more closely approximate the compositionof synovial fluid as compared to conventional viscosupplements and otherinjection materials used to treat joint diseases, disorders, andconditions, and are thus believed to better provide not only therequisite lubrication, cushioning, and protection of the joint, but alsothe components and pH level required for maintaining the healthybiological and physiological function of the entire joint system.

EXAMPLE Example 1 Treatment of Knee Osteoarthritis

A patient experiencing knee pain is diagnosed with knee osteoarthritis.The patient is administered a composition comprising a therapeuticallyeffective amount of TIMPs by injection directly into the synovial joint.The composition contains amniotic fluid that was harvested from anelective Caesarean section birth during week 37 of pregnancy. Painrelief is monitored using the Visual Analog Scale (VAS) and WesternOntario and McMaster Universities Arthritis Index (WOMAC) methods. Thecomposition is admininstered one or more times after the initialinjection, as needed, to further relieve pain.

It will be appreciated by those skilled in the art that changes could bemade to the embodiments described above without departing from the broadinventive concept thereof. It is understood, therefore, that thisinvention is not limited to the particular embodiments disclosed, but itis intended to cover modifications within the spirit and scope of thepresent invention as defined by the appended claims.

REFERENCES

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We claim:
 1. A method of treating a joint disease, disorder, orcondition in a subject in need thereof, the method comprising injectinga composition comprising an amniotic fluid comprising a therapeuticallyeffective amount of tissue inhibitors of matrix metalloproteinases(TIMPs) into the joint of the subject, thereby treating the jointdisease, disorder, or condition, wherein the amniotic fluid is obtainedfrom a pregnant female donor between week 28 and week 37 of gestation,wherein particulate matter in the amniotic fluid is substantiallyeliminated in the amniotic fluid prior to the composition being injectedinto the joint, and wherein a concentration of the TIMPs in a sample ofthe amniotic fluid is determined prior to injecting the composition intothe joint.
 2. The method according to claim 1, wherein the joint isselected from the group consisting of a synovial joint and acartilaginous joint.
 3. The method according to claim 1, wherein thejoint disease, disorder, or condition is associated with degradation ofcartilage by proteases.
 4. The method according to claim 3, wherein theproteases comprise matrix metalloproteinases.
 5. The method according toclaim 1, wherein the joint disease, disorder, or condition is selectedfrom the group consisting of joint inflammation, osteoarthritis,rheumatoid arthritis, and chondromalacia patellae.
 6. The methodaccording to claim 1, wherein the joint is a synovial joint.
 7. Themethod according to claim 1, wherein the joint is a knee joint.
 8. Themethod according to claim 1, wherein the amniotic fluid is obtained fromthe pregnant female donor during a Caesarean section procedure.
 9. Themethod according to claim 1, including determining that the amnioticfluid does not contain a therapeutically effective amount of the TIMPsand removing water from the amniotic fluid to enrich the amniotic fluidfor endogenous TIMPs whereby the enriched amniotic fluid includes thetherapeutically effective amount of the TIMPs.
 10. The method accordingto claim 1, wherein the joint disease, disorder, or condition isosteoarthritis of a synovial joint, wherein the osteoarthritis isassociated with degradation of articular cartilage by proteases, and thecomposition is injected into the synovial joint.
 11. The methodaccording to claim 10, wherein the proteases are matrixmetalloproteinases.
 12. The method according to claim 10, wherein thesynovial joint is a knee joint, wrist joint, shoulder joint, hip joint,elbow joint, or neck joint.
 13. The method according to claim 12,wherein the synovial joint is a knee joint.
 14. The method according toclaim 1, including determining that the amniotic fluid does not containa therapeutically effective amount of the TIMPs and increasing the TIMPsconcentration in the amniotic fluid whereby the enriched amniotic fluidincludes the therapeutically effective amount of the TIMPs.
 15. Themethod according to claim 1, wherein the amniotic fluid is processed byremoving water to enrich the amniotic fluid for endogenous TIMPs therebyproviding a processed amniotic fluid having a TIMPs concentrationselected from the group consisting of 10% w/v, 20% w/v, 30% w/v, 40%w/v, 50% w/v, 60% w/v, 70% w/v, 80% w/v, 90% w/v, 95% w/v and 99% w/v.16. A method of lubricating an osteoarthritic joint in a subject in needthereof, wherein the osteoarthritis of the joint is associated withdegradation of articular cartilage by proteases, the method comprisinginjecting into the joint a composition comprising an amniotic fluidcomprising a therapeutically effective amount of tissue inhibitors ofmatrix metalloproteinases, thereby lubricating the osteoarthritic jointwherein the amniotic fluid is obtained from a pregnant female donorbetween week 28and week 37of gestation, wherein particulate matter inthe amniotic fluid is substantially eliminated in the amniotic fluidprior to the composition being injected into the joint, and wherein aconcentration of the tissue inhibitors of matrix metalloproteinases in asample of the amniotic fluid is determined prior to injecting thecomposition into the joint, the concentration being about 10% w/v toabout 100% w/v.
 17. The method according to claim 16, wherein thesubject is in need of joint lubrication to provide pain-free jointmovement.
 18. A method of treating a joint disease, disorder, orcondition in a subject in need thereof, the method comprising obtainingamniotic fluid from a female donor at the time the female donorundergoes a Caesarean section delivery procedure, testing a sample ofthe amniotic fluid to determine a concentration of tissue inhibitors ofmatrix metalloproteinases (TIMPs) in the amniotic fluid, determiningwhether the amniotic fluid includes a therapeutically effective amountof TIMPs based upon the concentration of TIMPs in the amniotic fluid,minimizing particulate matter in the amniotic fluid, producing acomposition from the amniotic fluid, and injecting the compositioncomprising a therapeutically effective amount of tissue inhibitors ofTIMPs into the joint of the subject, thereby treating the joint disease,disorder, or condition, wherein, when it is determined that the amnioticfluid does not include the therapeutically effective amount of TIMPs,increasing the concentration of TIMPs in the amniotic fluid.
 19. Themethod according to claim 18, wherein the joint is a synovial joint andthe joint disease, disorder, or condition is osteoarthritis.
 20. Themethod according to claim 18, including adding recombinantly producedTIMPs to the amniotic fluid.